First synthesis of L-enantio-uracil dinucleotide.
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L-Enantio-uracil dinucleotide, which consists of two L-uridylic acids and one pyrophosphate, was synthesized for the first time in our laboratory. Benzolyated L-uridine was prepared by a steroselective glycosylation of silylated uracil with L-1-acetoxy-2,3,5-tri-O-benzoylribose (L-ABR). After deprotection, L-uridine was converted to L-UP4U by the treatment of L-UMP morpholidate with triethyammonium pyrophosphate (TEA-PPi). Spectral data of synthesized L-UP4U are given in a reference. All spectral data were identical with those of the D-enantiomer except the optical rotation. It showed a positive value compared to the D-enantiomer having a negative value. (+info)
Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex.
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The Trypanosoma brucei editosome catalyzes the maturation of mitochondrial mRNAs through the insertion and deletion of uridylates and contains at least 16 stably associated proteins. We examined physical and functional associations among these proteins using three different approaches: purification of complexes via tagged editing ligases TbREL1 and TbREL2, comprehensive yeast two-hybrid analysis, and coimmunoprecipitation of recombinant proteins. A purified TbREL1 subcomplex catalyzed precleaved deletion editing in vitro, while a purified TbREL2 subcomplex catalyzed precleaved insertion editing in vitro. The TbREL1 subcomplex contained three to four proteins, including a putative exonuclease, and appeared to be coordinated by the zinc finger protein TbMP63. The TbREL2 subcomplex had a different composition, contained the TbMP57 terminal uridylyl transferase, and appeared to be coordinated by the TbMP81 zinc finger protein. This study provides insight into the molecular architecture of the editosome and supports the existence of separate subcomplexes for deletion and insertion editing. (+info)
High-resolution crystal structures of ribonuclease A complexed with adenylic and uridylic nucleotide inhibitors. Implications for structure-based design of ribonucleolytic inhibitors.
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The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3',5'-ADP, 2',5'-ADP, 5'-ADP, U-2'-p and U-3'-p have been determined at high resolution. The structures reveal that each inhibitor binds differently in the RNase A active site by anchoring a phosphate group in subsite P1. The most potent inhibitor of all five, 5'-ADP (Ki = 1.2 microM), adopts a syn conformation (in contrast to 3',5'-ADP and 2',5'-ADP, which adopt an anti), and it is the beta- rather than the alpha-phosphate group that binds to P1. 3',5'-ADP binds with the 5'-phosphate group in P1 and the adenosine in the B2 pocket. Two different binding modes are observed in the two RNase A molecules of the asymmetric unit for 2',5'-ADP. This inhibitor binds with either the 3' or the 5' phosphate groups in subsite P1, and in each case, the adenosine binds in two different positions within the B2 subsite. The two uridilyl inhibitors bind similarly with the uridine moiety in the B1 subsite but the placement of a different phosphate group in P1 (2' versus 3') has significant implications on their potency against RNase A. Comparative structural analysis of the RNase A, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and human angiogenin (Ang) complexes with these and other phosphonucleotide inhibitors provides a wealth of information for structure-based design of inhibitors specific for each RNase. These inhibitors could be developed to therapeutic agents that could control the biological activities of EDN, ECP, and ANG, which play key roles in human pathologies. (+info)
Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape.
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The aim of this study was to investigate whether endogenous restrictions in oxygen supply are limiting for storage metabolism in developing oilseed rape (Brassica napus) seeds. Siliques were studied 30 d after flowering, when rapid lipid accumulation is occurring in the seeds. (a). By using microsensors, oxygen concentrations were measured within seeds and in the silique space between seeds. At ambient external oxygen (21% [v/v]) in the light, oxygen fell to 17% (v/v) between and 0.8% (v/v) within seeds. A step-wise reduction of the external oxygen concentration led within 2 h to a further decrease of internal oxygen concentrations, and a step-wise increase of the external oxygen concentration up to 60% (v/v) resulted in an increase in internal oxygen that rose to 30% (v/v) between and 8% (v/v) within seeds. (b). The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP, UTP, and the ATP to ADP and UTP to UDP ratios over the entire range from 0% to 60% (v/v) external oxygen. (c). To investigate metabolic fluxes in planta, 14C-sucrose was injected into seeds, which remained otherwise intact within their siliques. The increase in oxygen in the seeds was accompanied by a progressive increase in the rate of lipid (including triacylglycerol), protein and cell wall synthesis, and an increase in glycolytic flux over a range from sub- to superambient oxygen concentrations. In contrast to lipid synthesis, starch synthesis was not significantly increased at superambient oxygen levels. The levels of fermentation products such as lactate and glycerol-3P increased only at very low (0%-4% [v/v]) external oxygen concentrations. (d). When 14C-acetate or 14C-acetyl-coenzyme A (CoA) was injected into seeds, label incorporation into triacylglycerol progressively increased over the whole range of external oxygen concentrations from 0% to 60% (v/v). (e). Stimulation of lipid synthesis was accompanied by an increase in sugar levels and a decrease in the levels of hexose-phosphates and acetyl-CoA, indicating sucrose unloading and the use of acetyl-CoA as possible regulatory sites. (f). Increased lipid synthesis was also accompanied by an increase in the maximal activities of invertase and diacylglycerol acyltransferase. (g). The developmental shift from starch to lipid storage between 15 and 45 d after flowering was accompanied by an increase in the seed energy state. (h). The results show that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism and biosynthetic fluxes in growing rape seeds, affecting lipid synthesis more strongly than starch synthesis. The underlying mechanisms and implications for strategies to increase yield and storage product composition in oilseed crops are discussed. (+info)
Uridine 5'-polyphosphates (p4U and p5U) and uridine(5')polyphospho(5')nucleosides (Up(n)Ns) can be synthesized by UTP:glucose-1-phosphate uridylyltransferase from Saccharomyces cerevisiae.
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UTP:glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae can transfer the uridylyl moiety from UDP-glucose onto tripolyphosphate (P(3)), tetrapolyphosphate (P(4)), nucleoside triphosphates (p(3)Ns) and nucleoside 5'-polyphosphates (p(4)Ns) forming uridine 5'-tetraphosphate (p(4)U), uridine 5'-pentaphosphate (p(5)U) and dinucleotides, such as Ap(4)U, Cp(4)U, Gp(4)U, Up(4)U, Ap(5)U and Gp(5)U. Unlike UDP-glucose, UDP-galactose was not a UMP donor and ADP was not a UMP acceptor. This is the first example of an enzyme that may be responsible for accumulation of dinucleoside tetraphosphates containing two pyrimidine nucleosides in vivo. Occurrence of such dinucleotides in S. cerevisiae and Escherichia coli has been previously reported (Coste et al., J. Biol. Chem. 262 (1987) 12096-12103). (+info)
Brequinar potentiates 5-fluorouracil antitumor activity in a murine model colon 38 tumor by tissue-specific modulation of uridine nucleotide pools.
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Modulation of pyrimidine metabolism or the metabolic fate of 5-fluorouracil by a number of different agents has permitted a significant increase in the response rate to this agent, particularly for colorectal cancers. Brequinar, a noncompetitive inhibitor of mitochondrial dihydroorotate dehydrogenase has been shown to achieve a tumor-specific modulation of the therapeutic effect of 5-fluorouracil. A selective decrease of uridine nucleotide pools in Colon tumor 38 compared to normal tissues of C57/BL6 mice was observed after Brequinar administration. This effect was achieved with very low nontherapeutic doses of Brequinar (8 to 27% of the maximum tolerated dose in this model). Pretreatment with Brequinar 4 and 24 h prior to administration of [3H]fluorouracil significantly increased incorporation of the fluoropyrimidine into Colon 38 tumor RNA, while minimal effects were seen in normal tissues of C57/BL6 mice. Brequinar (15, 30, and 50 mg/kg) was administered 4 h prior to fluorouracil (85 mg/kg) on a weekly basis in Colon 38-bearing mice. All combinations potentiated 5-fluorouracil antitumor activity and the lowest dose of Brequinar (15 mg/kg) showed a reduced toxicity (weight loss) compared to the same dose of 5-fluorouracil as a single agent. When Brequinar preceded fluorouracil by 24 h, greater toxicity and less antitumor activity were observed. A comparison of the optimal Brequinar-fluorouracil regimen with a previously optimized N-(phosphonoacetyl)-L-aspartic acid-fluorouracil combination in Colon 38 tumor indicated that Brequinar-fluorouracil was more effective and less toxic. (+info)
Mutations in the RNA polymerase III subunit Rpc11p that decrease RNA 3' cleavage activity increase 3'-terminal oligo(U) length and La-dependent tRNA processing.
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Termination by RNA polymerase III (Pol III) produces RNAs whose 3' oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3' exonucleases and promotes their maturation. Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function. The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3' cleavage and to be important for termination. We used a La-dependent opal suppressor, tRNASerUGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression. Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3' cleavage activity and produce pre-tRNASerUGAM transcripts with elongated 3'-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNASerUGAM contains too few 3' Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants. The data indicate that Rpc11p limits RNA 3'-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast. (+info)
In Trypanosoma brucei RNA editing, band II enables recognition specifically at each step of the U insertion cycle.
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Trypanosome RNA editing is the posttranscriptional insertion and deletion of uridylate (U) residues, often to a massive extent, through cycles of cleavage, U addition or U removal, and ligation. These editing cycles are catalyzed by a complex that we purified to seven major proteins (bands I through VII). Here we analyze the role of band II using extracts of clonal band II RNA interference (RNAi) cell lines prepared by a rapid protocol that enables retention of activities that are lost during traditional extract preparation. By individually scoring each step of editing, we show that band II is critical for all steps of U insertion but is not important for any of the steps of U deletion or for their coordination into the U deletion cycle. This specificity supports the long- standing model that U-insertional and U-deletional activities are separated within the editing complex. Furthermore, by assaying the basic activities of the enzymes that catalyze the steps of U insertion, independent of their action in editing, we show that band II is not any of those enzymes. Rather, band II enables endonuclease action at authentic U insertion sites, terminal-uridylyl-transferase (TUTase) action at cleaved U insertion sites, and U-insertion-specific ligase (band V/IREL) action in the editing complex. Thus, band II facilitates each step of U insertion by providing proper RNA and/or protein recognition. We propose that band II (TbMP81) be called IRER, indicating its essential nature in U-insertional RNA editing recognition. (+info)